RESEARCH DESIGN AND METHODS

Adult male SD rats (Charles River Laboratories, Wilmington, MA), weighing 220–320 g, were studied after an overnight fast. Rats were housed at 22 ± 2°C, on a 12-h light-dark cycle and fed standard laboratory diet and water ad libitum prior to the study. After being anesthetized with pentobarbital sodium (50 mg/kg i.p.; Abbott Laboratories, North Chicago, IL), the rats were placed in a supine position on a heating pad to ensure euthermia and intubated to maintain a patent airway. The carotid artery and the jugular vein were cannulated with polyethylene tubing (PE-50; Fisher Scientific, Newark, DE) for arterial blood pressure monitoring, arterial blood sampling, and various infusions. After a 30- to 45-min baseline period to assure hemodynamic stability and a stable level of anesthesia, rats were studied under the following two protocols.

Protocol 1.

Five groups of rats were studied under this protocol (Fig. 1A). Group 1 rats received an intravenous infusion of regular insulin (3 mU/kg/min) for 120 min. Group 2 received systemic infusion of insulin (3 mU/kg/min) and PD123319 (AT2R blocker, 50 µg/kg/min, started at −5 min) for 120 min. Group 3 received a systemic infusion of insulin (3 mU/kg/min) for 120 min and PD123319 (50 µg/kg/min) 30 min after the initiation of insulin clamp. Group 4 received a bolus intravenous injection of losartan (AT1R blocker, 0.3 mg/kg) 5 min before the initiation of the insulin clamp. Arterial blood glucose was determined every 10 min using an Accu-Chek Advantage glucometer (Roche Diagnostics, Indianapolis, IN), and 30% dextrose (30% wt/vol) was infused at a variable rate to maintain blood glucose within 10% of basal (26,27). Group 5 rats received a saline infusion for 120 min.

Skeletal muscle microvascular blood volume (MBV), microvascular flow velocity (MFV), and microvascular blood flow (MBF) were determined using contrast-enhanced ultrasound, and femoral artery blood flow (FBF) was measured using a flow probe (VB series, 0.5 mm; Transonic Systems), as previously described (5,10,11,21). Plasma nitric oxide (NO) concentration was determined at the beginning and the end of insulin infusion, as described below. Rats then were killed and their gastrocnemius muscles freeze-clamped for later measurement of Akt phosphorylation using Western blotting, as previously described (26,27).

Protocol 2.

Two groups of rats were studied under this protocol (Fig. 1B). Group 1 received a continuous infusion of saline at 10 µL/min for 120 min. Group 2 received a systemic infusion of PD123319 (50 µg/kg/min) for 120 min. At 115 min, each rat received a bolus intravenous injection of 125I-insulin (1.5 µCi; Perkin Elmer, Boston, MA). Rats were killed at 120 min. Plasma and gastrocnemius were obtained for determination of 125I-insulin uptake.

Throughout the study, mean arterial blood pressure (MAP) and heart rate were monitored via a sensor connected to the carotid arterial catheter (Harvard Apparatus, Holliston, MA, and AD Instruments, Colorado Springs, CO). Pentobarbital sodium was infused at a variable rate to maintain steady levels of anesthesia and blood pressure throughout the study. Losartan and PD123319 were obtained from Sigma Chemicals (St. Louis, MO). Losartan was given as a bolus injection because of its longer plasma half-life (1.5–2.5 h for losartan and 6–9 h for its active metabolite), whereas PD123319 was given as continuous infusion because of its much shorter half-life (22 min). At the doses selected, neither losartan nor PD123319 significantly altered systemic blood pressure (21,28) but had a profound effect on basal microvascular perfusion (21). The plasma concentrations of losartan achieved were ~0.25 μg/mL (29) and of PD123319 were ~3 × 10−6 mol/L (30); values remained highly specific for AT1R and AT2R, respectively. The investigation conformed to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (publ. no. 85-23, revised 1996). The study protocols were approved by the animal care and use committee of the University of Virginia.

Measurement of plasma NO levels.

Plasma NO levels were measured using the 280i Nitric Oxide Analyzer (GE Analytical), according to the manufacturer’s instructions. In brief, ice-cold ethanol was added into plasma samples at a ratio of 2 to 1. The mixture was vortexed, kept at 0°C for 30 min, and then centrifuged at ~14,000 rpm for 5 min. The supernatant then was used for NO analysis.

Muscle 125I-insulin uptake.

In protocol 2 studies, each rat received a bolus intravenous injection of 1.5 μCi 125I-insulin 5 min prior to the end of the study. This tracer amount of insulin does not increase systemic insulin concentrations and thus can be used to track the uptake of native insulin. We chose this shorter duration of insulin exposure because of the short circulating half-life (<5 min) for intact insulin (31). At the end of the experiment, a blood sample was collected and each rat then was flushed with 120 mL ice-cold saline (10 mL/min) via the carotid artery catheter. Gastrocnemius muscles were dissected from the right hindlimbs. Protein-bound 125I in blood and muscle samples were precipitated with 30% trichloroacetic acid and was radioactivity measured. Skeletal muscle insulin uptake was calculated using the following formula: muscle insulin uptake = 125I-insulin in muscle (dpm/g dry wt/5 min)/blood 125I-insulin (dpm/mL).

Statistical analysis.

All data are presented as means ± SEM. Statistical analyses were performed with SigmaStat 3.1.1 software (Systat Software), using one-way ANOVA or one-way repeated-measures ANOVA with post hoc Holm-Sidak or Dunn analysis, where appropriate. A P value <0.05 was considered statistically significant.

RESULTS

We have previously demonstrated that basal AT1R and AT2R regulate muscle microvascular volume and glucose use (21). Because the microvasculature plays an important role in insulin delivery and substrate exchange between the plasma and interstitial compartments, we first examined whether changes in AT1R and AT2R activities altered insulin-mediated glucose disposal. As shown in Fig. 2, injection of losartan had no effect on insulin-mediated glucose disposal. On the other hand, PD123319 infusion, given either immediately before or 30 min after the initiation of insulin infusion, promptly attenuated insulin-stimulated whole-body glucose disposal, and this effect was maintained during the entire 120 or 90 min of PD123319 infusion (P < 0.02, ANOVA). At steady state, AT2R blockade decreased the whole-body glucose disposal rate by ~30% (P = 0.02 for both PD123319 groups). In saline-only rats, the microvascular parameters did not change significantly during the course of the study.

AT2R antagonism attenuates insulin-mediated whole-body glucose disposal. Each rat received 2 h of insulin infusion in the absence or presence of either PD123319 infusion (50 μg/kg/min, started at time −5 or 30 min) or losartan injection (0.3 mg/kg i.v. at time −5 min). n = 8–16. *P < 0.02 (ANOVA).

We have shown that basal AT1R tone restricts skeletal muscle MBV, whereas basal AT2R activity increases muscle MBV (21). We next examined whether selective stimulation of the ANG II subtype receptors by endogenous ANG II regulate insulin-mediated microvascular recruitment. Figure 3 shows the microvascular responses to insulin infusion. Insulin infusion raised plasma insulin concentrations from 99 ± 15 pmol/L to 685 ± 151 pmol/L (n = 5, P < 0.02). As expected, insulin increased both MBV and MBF without altering MFV (Fig. 3). The addition of PD123319 to insulin infusion promptly inhibited this insulin-mediated increase in MBV and MBF and significantly increased the MFV (Fig. 4). When PD123319 was given before insulin infusion, insulin-induced increases in muscle MBV and MBF were completely prevented. On the other hand, infusion of PD123319 after insulin had already significantly recruited muscle microvasculature (i.e., 30 min after insulin infusion) abrogated the insulin effects. These changes did not seem to be secondary to changes in blood pressure or total blood flow because both MAP and FBF remained stable during the study (Table 1). Injection of losartan 5 min prior to the initiation of insulin promptly increased muscle MBV within 30 min, and the extent of the increase (2.4- to 3.2-fold) was quite similar to what we previously observed using losartan alone (21). The increase in MBV lasted for the entire 120 min. Muscle MFV did not change in the first 90 min but decreased at 120 min. As a result, muscle MBF increased promptly at 30 min, remained elevated at 60 min, and trended down afterward (Fig. 5). Though MAP did not change, FBF decreased by nearly 30% (P < 0.01) (Table 1).

The rapid attenuation of insulin-stimulated whole-body glucose disposal and muscle microvascular recruitment by PD123319 prompted us to further examine the impact of ANG II receptor stimulation by endogenous ANG II on the insulin effects on the vasculature (NO production) and muscle (Akt phosphorylation). Both insulin and losartan increase microvascular recruitment via a NO-dependent pathway (6,21). As shown in Fig. 6A, 2 h of insulin infusion increased plasma NO levels by nearly twofold (P < 0.01). Concurrent PD123319 infusion decreased insulin-mediated increases in plasma NO levels by 37% (P < 0.04) to levels that were not significantly different from the saline controls. On the other hand, losartan injection had no significant impact on insulin-stimulated NO production. Figure 6B shows insulin-stimulated Akt phosphorylation in the skeletal muscle. Similar to the pattern of plasma NO levels, insulin significantly increased muscle Akt phosphorylation, and this effect was suppressed back to the saline control level when PD123319 infusion was superimposed on insulin infusion. Again, losartan had no effect on insulin-stimulated Akt phosphorylation in muscle. Muscle endothelial NO synthase (eNOS) phosphorylation did not differ among all four groups (Fig. 6C).

DISCUSSION

In humans, ANG II infusion acutely enhances insulin-stimulated muscle glucose disposal (18–20). This was suggested to be attributable to enhanced tissue perfusion, although evidence for this was not available. We recently have found that both the AT1R and AT2R exert potent basal tonic effects on microvasculature in skeletal muscle (21). Here, we show that both AT1R and AT2R regulate insulin’s microvascular and metabolic actions in muscle. Although the blockade of AT1R significantly increased muscle microvascular perfusion, it did not further enhance insulin-stimulated whole-body glucose disposal, NO production, and muscle Akt phosphorylation. In contrast, AT2R antagonism promptly attenuated muscle metabolic responses to insulin, which was associated with decreased muscle insulin uptake and insulin-mediated NO production and microvascular recruitment. Although we previously have demonstrated that dual blockade of both AT1R and AT2R had neutral effects on muscle microvascular recruitment and basal glucose extraction, whether dual blockade alters microvascular and muscle insulin sensitivity is unclear. In addition, our observations may represent a combined peripheral and central effect because it is likely that both losartan and PD123319 could exert central effects via either penetrating the blood-brain barrier or acting on the blood vessels in the central nervous system (28). An alternative is to administrate these compounds locally into the microvasculature via the epigastric artery (32). However, this would require abdominal incision, which further stresses the animals, and prolonged infusions would likely also raise systemic concentrations.

Similar to the previous reports (8,12,33–36), insulin potently increased muscle MBV by 58–75%. Injection of losartan prior to the initiation of insulin infusion increased muscle MBV by approximately two- to threefold (Fig. 5), which is similar to what we observed previously with losartan alone (21). Thus, there seems to be no additive effects of losartan and insulin on muscle MBV. It is likely that the large increase in MBV achieved with the losartan injection may have masked the insulin effect. The large increase in muscle MBV at 30 min without an increase in FBF likely reflects a shift in flow distribution from nonnutritive to nutritive microvascular beds (37). Of interest, MBF trended down after 90 min, which was likely secondary to the gradual decline in FBF and a decrease in MFV at 120 min. Whether these findings are caused by either losartan alone or losartan plus insulin remain to be clarified because MFV and MBF were not determined in our previous study using losartan alone (21). Compared with increases induced by insulin alone (Fig. 3), the increases in muscle MBV induced by losartan and insulin combination was only significantly higher at 30 min (P < 0.03), and increases in muscle MBF were not statistically different between the two groups. Thus, it is not surprising that the addition of losartan to insulin did not further increase muscle insulin action beyond what we saw with insulin alone because insulin-mediated microvascular recruitment is tightly coupled with insulin’s metabolic action.

AT2R has been associated with vasodilation in large capacitance vessels, resistance arterioles, as well as the microvasculature (21,23,38–41), likely via the bradykinin-NO-cGMP signaling cascade because the AT2R antagonist PD123319, the bradykinin receptor 2 antagonist icatibant, and the NO synthase inhibitor l-NAME each independently blocks this action (25,38,42,43). We previously have reported that skeletal muscle precapillary arterioles are a major site of AT2R action in vivo because blockade of the AT1R with losartan causes a threefold increase in muscle MBV, whereas blockade of the AT2R renders an ~80% reduction in muscle MBV (21). Our current results confirmed that AT2R activity also plays an important role in the regulation of insulin’s microvascular and metabolic responses in muscle as well. Indeed, PD123319 caused a prompt decrease in insulin-mediated glucose disposal, which was associated with abrogation of insulin-stimulated muscle microvascular recruitment, NO production and muscle Akt phosphorylation, and muscle insulin uptake. Thus, AT2Rs are important regulators of both basal microvascular tone and insulin delivery/action in muscle via its effects on the microvasculature, which is consistent with the current understanding that the regulation of microvascular insulin delivery is a major rate-limiting step in skeletal muscle insulin action (2,44–47).

It is important to distinguish between the acute effects we observed and what might be going on when the RAS already is at a higher level as occurs in obesity and diabetes. Increased muscle microvascular perfusion induced by muscle contraction is clearly associated with increased muscle insulin uptake even in the presence of high plasma free fatty acids (10,11). In humans, simple obesity blunts insulin- and mixed-meal–stimulated muscle microvascular recruitment (9,35). Likewise, in obese Zucker rats, an animal model of metabolic syndrome and insulin resistance, basal skeletal muscle MBV is decreased, which is coupled with impaired insulin-mediated glucose disposal and microvascular recruitment (48). Insulin uptake has not been assessed in this model. However, treatment of the Zucker diabetic fatty rats with the ACE inhibitor quinapril restores insulin’s microvascular action and improves insulin-mediated whole-body glucose disposal (49). Because the cardiovascular RAS is upregulated in diabetes, which has been implicated in the development of diabetic cardiovascular complications (7,13,24,50), blockade of the AT1R and/or activation of the AT2R may help to improve insulin delivery and sensitivity and potentially improve the cardiovascular outcomes in patients with diabetes. Although the current study revealed important modulatory roles of ANG II subreceptors on insulin action under normal physiology, whether the current findings can be extrapolated to the insulin-resistant state is unclear and requires additional studies.

We previously have shown in humans that physiologic hyperinsulinemia increases muscle MBF solely by increasing MBV without changing the MFV (8,35,36). Our current study in rodents confirmed this lack of change in MFV after insulin stimulation as well (Figs. 3 and 4). Although the addition of losartan to the insulin infusion caused a decrease in MFV at 120 min, superimposing the PD123319 infusion not only promptly attenuated insulin-stimulated MBV but it also increased muscle MFV. Because there was no change in total FBF, this increase in MFV was likely secondary to the acute decrease in MBV.

Our finding that insulin increased plasma NO levels is consistent with many previous reports demonstrating that insulin causes vasodilation via the phosphatidylinositol 3-kinase/Akt/eNOS pathway (2,4–6,51). We previously have demonstrated that losartan treatment led to microvascular recruitment via the AT2R-NO–dependent mechanism because l-NAME treatment abolished losartan-induced increases in MBV and glucose extraction (21). The increase in plasma NO levels seen with combined losartan and insulin treatment most likely reflects increased release of NO from the endothelium secondary to endogenous ANG II stimulation of the AT2R/B2R/eNOS pathway and insulin stimulation of the phosphatidylinositol 3-kinase/Akt/eNOS pathway. The NO release is less likely from skeletal muscle cells, because they express predominantly AT1R (52), which activates NADPH oxidase, increases reactive oxygen species, and results in decreased NO bioavailability. In the current study, NO production did not further increase with the combined losartan and insulin treatment, which is consistent with the findings that the increases in MBV and MBF were not statistically different between the two groups.

According to the Fick principle, increases in the endothelial exchange surface area between the plasma and the interstitial compartments in muscle could significantly increase the delivery of insulin to the muscle microvasculature and facilitate its transfer to the interstitial space and enhance its metabolic action. Our observation that insulin-stimulated glucose disposal and muscle Akt phosphorylation, two major indices of insulin metabolic actions in muscle, closely parallel the changes in insulin-mediated microvascular recruitment and NO production, and muscle insulin uptake strongly supports this concept. Indeed, PD123319 superimposing on insulin infusion decreased all these major end points in the current study. The abrogation of insulin-induced microvascular recruitment with PD123319 infusion most likely resulted in a decreased insulin delivery and uptake in the muscle because it is in the microvasculature where insulin uptake takes place and decreased exchange surface area could significantly decrease muscle insulin uptake. The decreases in NO production and Akt phosphorylation with PD123319 infusion are likely secondary to decreased stimulation of the AT2R by ANG II and insulin receptor by insulin on the endothelium (for NO) and decreased insulin delivery to the muscle (for p-Akt).

In conclusion, both AT1Rs and AT2Rs regulate insulin’s microvascular and metabolic actions in muscle. Our current findings strongly suggest that ANG receptors affect insulin sensitivity in vivo via modulating microvascular perfusion. This is an important finding because changes in the microvascular endothelial surface area significantly affects insulin delivery to and action in muscle (2). Additional studies are needed to define the underlying molecular mechanisms. Although AT1R activity restrains muscle metabolic responses to insulin via decreased muscle microvascular recruitment and insulin delivery, AT2R activity is required for normal muscle microvascular and metabolic responses to insulin. Thus, pharmacologic manipulation aimed at increasing the AT2R-to-AT1R activity ratio may afford potential to improve muscle insulin sensitivity and glucose metabolism and to reduce the cardiovascular complications associated with diabetes and insulin resistance.

ACKNOWLEDGMENTS

This work was supported by American Diabetes Association grants 1-11-CR-30 and 9-09-NOVO-11 (to Z.L.), National Institutes of Health Grant R01-HL-094722 (to Z.L.), and National Natural Science Foundation of China Grant 81070632 (to W.W.).

No potential conflicts of interest relevant to this article were reported.

. Direct administration of insulin into skeletal muscle reveals that the transport of insulin across the capillary endothelium limits the time course of insulin to activate glucose disposal. Diabetes2008;57:828–835pmid:18223011